Abstract
Vaccinia DNA topoisomerase specifically binds and forms a covalent adduct at DNA sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. The molecular interactions that contribute to recognition of the CCCTT motif in a synthetic DNA substrate have been examined using modification interference, modification protection, and analog substitution techniques. We report that topoisomerase makes contact with guanine nucleotide bases of the pentamer motif complementary strand (3'GGGAA) within the major groove of the DNA helix and that alteration of the binding surface by chemical modification is deleterious to the interaction. Additional contacts are made with guanine residues located outside the pentamer element. The enzyme is unable to form a covalent adduct with synthetic RNA substrates. Analysis of the cleavage of DNA duplexes containing 2'OMe sugars suggests that the inability of the vaccinia topoisomerase to cleave either an RNA duplex or an RNA:DNA hybrid can be accounted for by the interfering effects of a 2' sugar substituent at two or more sites within the pentamer. Interaction with the sugar at the +2T nucleotide appears to be the most critical, as judged by the effects of single sugar substitutions.
Highlights
Vaccinia DNA topoisomerase binds and A requirement for covalent complex formation between formsacovalentadductatDNA sites containinga vaccinia topoisomerase and DNA is that thCeCCTT sequence conserved sequence element 6’(C/T)CCTTinJthe scis- be in duplex form (4)
Analysis of the The vaccinia topoisomerase, upon binding to a duplex DNA cleavage of DNA duplexes containing 2’OMe sugars containing a CCCTT motif, protects the region suggests that the inability of the vaccinia topoisomera-round the site of covalent adduct formation from DNase I ase to cleave either an RNA duplex or an RNA:DNA digestion (5).The DNase footprint spans both sides of the hybrid can be accounted for by the interferinefgfects cleavage site, from +13 to -13 on thescissile strand (+1being of a 2’ sugar substituent at two or more sites within the site of cleavage) and from +13 to -9 on the noncleaved the pentamer
Valent adduct formation(5).the margins of the nuclease footprint extend beyond the minimal essential positions for strand cleavage defined by DNAdeletion and Topological transactions executed by the eukaryotic type I DNA topoisomerases can be broken down into a series of discrete partial reactions (1).A single catalytic cycle entails: (i) noncovalent binding of enzymeto duplex DNA; (ii) scission of one DNA strand with concomitant formationof a covalent protein-DNA adduct; (iii) strand passage or free rotation of the cleaved strand; (iv) religation across the bond originally broken, and; (v) dissociation of enzyme from the DNA
Summary
From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021. The size of the footprint is greater than the11bp’ of nucleotide appears to be the mcorsitical, as judged by duplex DNA that constitute the “minimal” substrate for cothe effects of single sugar substitutions. Vaccinia topoisomerase binds and forms a covalent adduct at DNA sites containing the conserved sequence 5’(C/T)CCTTI (2). The DNA mutational analyses and nuclease footprinting experiments confirm the sequence specificity of the topoisomerase-DNA interaction, they do not illuminate the pertinent principles of site recognition. This issue is addressed in the presentstudy using a combination of modification interference, modification protection, andanalog substitution techniques. Residue (designated position +1)is linked to Tyr-274 of the enzyme via a 3‘ phosphodiester bond (3)
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